Polynucleotides encoding prostatic growth factor and process for producing prostatic growth factor polypeptides

ABSTRACT

The present invention relates to polynucleotides encoding human PGF polypeptides, variant polynucleotides encoding analog polypeptides, and variant polynucleotides useful as probes for polynucleotides encoding human PGF polypeptides. Also provided is a procedure for producing such polypeptides by recombinant techniques.

This application is entitled to the benefits of 35 U.S.C. §120 forpriority based on PCT/US94/14578, filed Dec. 15, 1994.

This invention relates to newly identified polynucleotides, polypeptidesencoded by such polynucleotides, the use of such polynucleotides andpolypeptides, as well as the production of such polynucleotides andpolypeptides. More particularly, the polypeptide of the presentinvention is a prostatic growth factor which is sometimes hereinafterreferred to as "PGF".

This invention relates to a polynucleotide and polypeptide moleculeswhich are structurally and functionally related to TGF-β. Thetransforming growth factor-beta family of peptide growth factorsincludes five members, termed TGF-β1 through TGF-β5, all of which formhomo-dimers of approximately 25 kd. The TGF-β family belongs to alarger, extended super family of peptide signaling molecules thatincludes the Muellerian inhibiting substance (Cate, R. L. et al., Cell,45:685-698 (1986)), decapentaplegic (Padgett, R. W. et al., Nature,325:81-84 (1987)), bone morphogenic factors (Wozney, J. M. et al.,Science, 242:1528-1534 (1988)), vg1 (Weeks, D. L., and Melton, D. A.,Cell, 51:861-867 (1987)), activins (Vale, W. et al., Nature, 321:776-779(1986)), and inhibins (Mason, A. J. et al., Nature, 318:659-663 (1985)).These factors are similar to TGF-β in overall structure, but share onlyapproximately 25% amino acid identity with the TGF-β proteins and witheach other. All of these molecules are thought to play an importantroles in modulating growth, development and differentiation. The proteinof the present invention, PGF, retains the seven cysteine residuesconserved in the C-terminal, active domain of TGF-β.

TGF-β was originally described as a factor that induced normal ratkidney fibroblasts to proliferate in soft agar in the presence ofepidermal growth factor (Roberts, A. B. et al., PNAS USA, 78:5339-5343(1981)). TGF-β has subsequently been shown to exert a number ofdifferent effects in a variety of cells. For example, TGF-β can inhibitthe differentiation of certain cells of mesodermal origin (Florini, J.R. et al., J. Biol. Chem., 261:1659-16513 (1986)), induced thedifferentiation of others (Seyedine, S. M. et al., PNAS USA,82:2267-2271 (1985)), and potently inhibit proliferation of varioustypes of epithelial cells, (Tucker, R. F., Science, 226:705-707 (1984)).This last activity has lead to the speculation that one importantphysiologic role for TGF-β is to maintain the repressed growth state ofmany types of cells. Accordingly, cells that lose the ability to respondto TGF-β are more likely to exhibit uncontrolled growth and to becometumorigenic. Indeed, the cells lack certain tumors such asretinoblastomas lack detectable TGF-β receptors at their cell surfaceand fail to respond to TGF-β, while their normal counterparts expressself-surface receptors in their growth is potently inhibited by TGF-β(Kim Chi, A. et al., Science, 240:196-198 (1988)).

More specifically, TGF-β1 stimulates the anchorage-independent growth ofnormal rat kidney fibroblasts (Robert et al., PNAS USA, 78:5339-5343(1981)). Since then it has been shown to be a multi-functional regulatorof cell growth and differentiation (Sporn et al., Science, 233:532-534(1986)) being capable of such diverse effects of inhibiting the growthof several human cancer cell lines (Roberts et al., PNAS-USA, 82:119-123(1985)), mouse keratinocytes, (Coffey et al., Cancer RES., 48:1596-1602(1988)), and T and B lymphocytes (Kehrl et al., J. Exp. Med.,163:1037-1050 (1986)). It also inhibits early hematopoietic progenitorcell proliferation (Goey et al., J. Immunol., 143:877-880 (1989)),stimulates the induction of differentiation of rat muscle mesenchymalcells and subsequent production of cartilage-specific macro molecules(Seyedine et al., J. Biol. Chem., 262:1946-1949 (1986)), causesincreased synthesis and secretion of collagen (Ignotz et al., J. Biol.Chem., 261:4337-4345 (1986)), stimulates bone formation (Noda et al.,Endocrinology, 124:2991-2995 (1989)), and accelerates the healing ofincision wounds (Mustoe et al., Science, 237:1333-1335 (1987)).

Further, TGF-β1 stimulates formation of extracellular matrix moleculesin the liver and lung. When levels of TGF-β1 are higher than normal,formation of fiber occurs in the extracellular matrix of the liver andlung which can be fatal. High levels of TGF-β1 occur due to chemotherapyand bone marrow transplant as an attempt to treat cancers, eg. breastcancer.

A second protein termed TGF-β2 was isolated from several sourcesincluding demineralized bone, a human prostatic adenocarcinoma cell line(Ikeda et al., Bio. Chem., 26:2406-2410 (1987)). TGF-β2 shared severalfunctional similarities with TGF-β1. These proteins are now known to bemembers of a family of related growth modulatory proteins includingTGF-β3 (Ten-Dijke et al., PNAS-USA, 85:471-4719 (1988)), Muellerianinhibitory substance and the inhibins. Due to amino acid sequencehomology, it is thought that the PGF polypeptide of the presentinvention is also a member of this family of related growth modulatoryproteins. However, to date, this polypeptide has only been found by theinventors to be present in the prostate.

In accordance with one aspect of the present invention, there isprovided a novel mature polypeptide which is PGF, as well asbiologically active and diagnostically or therapeutically usefulfragments, analogs and derivatives thereof. The polypeptide of thepresent invention is of human origin.

In accordance with another aspect of the present invention, there areprovided isolated nucleic acid molecules encoding human PGF, includingmRNAs, DNAs, cDNAs, genomic DNAs as well as analogs and biologicallyactive and diagnostically or therapeutically useful fragments andderivatives thereof.

In accordance with yet a further aspect of the present invention, thereis provided a process for producing such polypeptide by recombinanttechniques comprising culturing recombinant prokaryotic and/oreukaryotic host cells, containing a human PGF nucleic acid sequence,under conditions promoting expression of said protein and subsequentrecovery of said protein..

In accordance with yet a further aspect of the present invention, thereis provided a process for utilizing such polypeptide, or polynucleotideencoding such polypeptide for therapeutic purposes, for example, toinhibit prostate cancer, stimulate tissue regeneration and to promotewound healing.

In accordance with yet a further aspect of the present invention, thereare provided antibodies against such polypeptides.

In accordance with yet another aspect of the present invention, thereare provided antagonists to such polypeptides, which may be used toinhibit the action of such polypeptides, for example, in the treatmentof PGF-dependent tumors.

In accordance with yet a further aspect of the present invention, thereare also provided nucleic acid probes comprising nucleic acid moleculesof sufficient length to specifically hybridize to human PGF sequences.

In accordance with still another aspect of the present invention, thereare provided diagnostic assays for detecting diseases related to theunder-expression and over-expression of the PGF polypeptide andmutations in the nucleic acid sequences encoding such polypeptide.

In accordance with yet a further aspect of the present invention, thereis provided a process for utilizing such polypeptides, orpolynucleotides encoding such polypeptides, for in vitro purposesrelated to scientific research, synthesis of DNA and manufacture of DNAvectors.

These and other aspects of the present invention should be apparent tothose skilled in the art from the teachings herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings are illustrative of embodiments of the inventionand are not meant to limit the scope of the invention as encompassed bythe claims.

FIG. 1 depicts the cDNA sequence (SEQ ID NO:1) and corresponding deducedamino acid sequence (SEQ ID NO:2) of PGF. The standard one-letterabbreviations for amino acids are used. Sequencing was performed using a373 Automated DNA sequencer (Applied Biosystems, Inc.). Sequencingaccuracy is predicted to be greater than 97% accurate.

FIG. 2 is an illustration of comparative amino acid homology between theamino acid sequence of PGF and of two other proteins. The sequences arerepresented by one-letter amino acid codes and are show in relativealignment to one another. The first line is the relevant portion of PGF(SEQ ID NO:2) as compared to lines two and three, OP-1 (human osteogenicprotein, SEQ ID NO:3) and Vg-1 (X. laevis vegetal hemisphere Vg-1protein precursor, SEQ ID NO:4), respectively. Although gaps may beshown in the amino acid sequences in the comparative illustration ofFIG. 2, for convenience SEQ ID NOS:3 and 4 are placed in the sequencelisting as continuous sequences.

In accordance with an aspect of the present invention, there is providedan isolated nucleic acid (polynucleotide) which encodes for the maturepolypeptide having the deduced amino acid sequence of FIG. 1 or for themature polypeptide encoded by the cDNA of the clone deposited as ATCCDeposit 75902 on Sep. 28, 1994.

The ATCC number referred to above is directed to a biological depositwith the ATCC, 10801 University Boulevard, Manassas, Va. 20110-2209.Since the strain referred to is being maintained under the terms of theBudapest Treaty, it will be made available to a patent office signatoryto the Budapest Treaty.

A polynucleotide encoding a polypeptide of the present invention may beobtained from human fetal spleen, prostate and 6 week old embryo. Thepolynucleotide of this invention was discovered in a cDNA libraryderived from human prostate. It is structurally related to the TGF-βfamily. It contains an open reading frame encoding a protein ofapproximately 276 amino acid residues of which approximately the first15 amino acids residues are the putative leader sequence such that themature protein comprises 261 amino acids. The protein exhibits thehighest degree of homology to human osteogenic protein 1 (OP-1) with 33%identity and 57% similarity over a 45 amino acid stretch. Thepolypeptide contains the seven conserved cysteine amino acidscharacteristic of the TGF-β family members C-terminal domain.

The polynucleotide of the present invention may be in the form of RNA orin the form of DNA, which DNA includes cDNA, genomic DNA, and syntheticDNA. The DNA may be double-stranded or single-stranded, and if singlestranded may be the coding strand or non-coding (anti-sense) strand. Thecoding sequence which encodes the mature polypeptide may be identical tothe coding sequence shown in FIG. 1 or that of the deposited clone ormay be a different coding sequence which coding sequence, as a result ofthe redundancy or degeneracy of the genetic code, encodes the samemature polypeptide as the DNA of FIG. 1 or the deposited cDNA.

The polynucleotide which encodes for the mature polypeptide of FIG. 1 orfor the mature polypeptide encoded by the deposited cDNA may include:only the coding sequence for the mature polypeptide; the coding sequencefor the mature polypeptide (and optionally additional coding sequence)and non-coding sequence, such as introns or non-coding sequence 5'and/or 3' of the coding sequence for the mature polypeptide.

Thus, the term "polynucleotide encoding a polypeptide" encompasses apolynucleotide which includes only coding sequence for the polypeptideas well as a polynucleotide which includes additional coding and/ornon-coding sequence.

The present invention further relates to variants of the hereinabovedescribed polynucleotides which encode for fragments, analogs andderivatives of the polypeptide having the deduced amino acid sequence ofFIG. 1 or the polypeptide encoded by the cDNA of the deposited clone.The variant of the polynucleotide may be a naturally occurring allelicvariant of the polynucleotide or a non-naturally occurring variant ofthe polynucleotide.

Thus, the present invention includes polynucleotides encoding the samemature polypeptide as shown in FIG. 1 or the same mature polypeptideencoded by the cDNA of the deposited clone as well as variants of suchpolynucleotides which variants encode for a fragment, derivative oranalog of the polypeptide of FIG. 1 or the polypeptide encoded by thecDNA of the deposited clone. Such nucleotide variants include deletionvariants, substitution variants and addition or insertion variants.

As hereinabove indicated, the polynucleotide may have a coding sequencewhich is a naturally occurring allelic variant of the coding sequenceshown in FIG. 1 or of the coding sequence of the deposited clone. Asknown in the art, an allelic variant is an alternate form of apolynucleotide sequence which may have a substitution, deletion oraddition of one or more nucleotides, which does not substantially alterthe function of the encoded polypeptide.

The polynucleotides of the present invention may also have the codingsequence fused in frame to a marker sequence which allows forpurification of the polypeptide of the present invention. The markersequence may be a hexahistidine tag supplied by a pQE-9 vector toprovide for purification of the mature polypeptide fused to the markerin the case of a bacterial host, or, for example, the marker sequencemay be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells,is used. The HA tag corresponds to an epitope derived from the influenzahemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).

The present invention further relates to polynucleotides which hybridizeto the hereinabove-described sequences if there is at least 50% andpreferably 70% identity between the sequences. The present inventionparticularly relates to polynucleotides which hybridize under stringentconditions to the hereinabove-described polynucleotides. As herein used,the term "stringent conditions" means hybridization will occur only ifthere is at least 95% and preferably at least 97% identity between thesequences. The polynucleotides which hybridize to the hereinabovedescribed polynucleotides in a preferred embodiment encode polypeptideswhich retain substantially the same biological function or activity asthe mature polypeptide encoded by the cDNA of FIG. 1 or the depositedcDNA.

The deposit(s) referred to herein will be maintained under the terms ofthe Budapest Treaty on the International Recognition of the Deposit ofMicro-organisms for purposes of Patent Procedure. These deposits areprovided merely as convenience to those of skill in the art and are notan admission that a deposit is required under 35 U.S.C. §112. Thesequence of the polynucleotides contained in the deposited materials, aswell as the amino acid sequence of the polypeptides encoded thereby, areincorporated herein by reference and are controlling in the event of anyconflict with any description of sequences herein. A license may berequired to make, use or sell the deposited materials, and no suchlicense is hereby granted.

The present invention further relates to a polypeptide which has thededuced amino acid sequence of FIG. 1 or which has the amino acidsequence encoded by the deposited cDNA, as well as fragments, analogsand derivatives of such polypeptide.

The terms "fragment," "derivative" and "analog" when referring to thepolypeptide of FIG. 1 or that encoded by the deposited cDNA, means apolypeptide which retains essentially the same biological function oractivity as such polypeptide. Thus, an analog includes a proproteinwhich can be activated by cleavage of the proprotein portion to producean active mature polypeptide.

The polypeptide of the present invention may be a recombinantpolypeptide, a natural polypeptide or a synthetic polypeptide,preferably a recombinant polypeptide.

The fragment, derivative or analog of the polypeptide of FIG. 1 or thatencoded by the deposited cDNA may be (i) one in which one or more of theamino acid residues are substituted with a conserved or non-conservedamino acid residue (preferably a conserved amino acid residue) and suchsubstituted amino acid residue may or may not be one encoded by thegenetic code, or (ii) one in which one or more of the amino acidresidues includes a substituent group, or (iii) one in which the maturepolypeptide is fused with another compound, such as a compound toincrease the half-life of the polypeptide (for example, polyethyleneglycol). Such fragments, derivatives and analogs are deemed to be withinthe scope of those skilled in the art from the teachings herein.

The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

The term "isolated" means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

The present invention also relates to vectors which includepolynucleotides of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques.

Host cells are genetically engineered (transduced or transformed ortransfected) with the vectors of this invention which may be, forexample, a cloning vector or an expression vector. The vector may be,for example, in the form of a plasmid, a viral particle, a phage, etc.The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying the PGF genes. The culture conditions, suchas temperature, pH and the like, are those previously used with the hostcell selected for expression, and will be apparent to the ordinarilyskilled artisan.

The polynucleotides of the present invention may be employed forproducing polypeptides by recombinant techniques. Thus, for example, thepolynucleotide may be included in any one of a variety of expressionvectors for expressing a polypeptide. Such vectors include chromosomal,nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40;bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectorsderived from combinations of plasmids and phage DNA, viral DNA such asvaccinia, adenovirus, fowl pox virus, and pseudorabies. However, anyother vector may be used as long as it is replicable and viable in thehost.

The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease site(s) by procedures known in theart. Such procedures and others are deemed to be within the scope ofthose skilled in the art.

The DNA sequence in the expression vector is operatively linked to anappropriate expression control sequence(s) (promoter) to direct mRNAsynthesis. As representative examples of such promoters, there may bementioned: LTR or SV40 promoter, the E. coli. lac or trp, the phagelambda P_(L) promoter and other promoters known to control expression ofgenes in prokaryotic or eukaryotic cells or their viruses. Theexpression vector also contains a ribosome binding site for translationinitiation and a transcription terminator. The vector may also includeappropriate sequences for amplifying expression.

In addition, the expression vectors preferably contain one or moreselectable marker genes to provide a phenotypic trait for selection oftransformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or such as tetracycline orampicillin resistance in E. coli.

The vector containing the appropriate DNA sequence as hereinabovedescribed, as well as an appropriate promoter or control sequence, maybe employed to transform an appropriate host to permit the host toexpress the protein.

As representative examples of appropriate hosts, there may be mentioned:bacterial cells, such as E. coli, Streptomyces, Salmonella typhimurium;fungal cells, such as yeast; insect cells such as Drosophila S2 and Sf9;animal cells such as CHO, COS or Bowes melanoma; adenoviruses; plantcells, etc. The selection of an appropriate host is deemed to be withinthe scope of those skilled in the art from the teachings herein.

More particularly, the present invention also includes recombinantconstructs comprising one or more of the sequences as broadly describedabove. The constructs comprise a vector, such as a plasmid or viralvector, into which a sequence of the invention has been inserted, in aforward or reverse orientation. In a preferred aspect of thisembodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art, and are commercially-available. The following vectorsare provided by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen),pBS, pD10, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a,pNH18A, pNH46A (Stratagene); pTRC99a, pKK223-3, pKK233-3, pDR540, pRIT5(Pharmacia). Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene)pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid orvector may be used as long as they are replicable and viable in thehost.

Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are PKK232-8 and PCM7. Particular namedbacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P_(R), P_(L)and trp. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art.

In a further embodiment, the present invention relates to host cellscontaining the above-described constructs. The host cell can be a highereukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell,such as a yeast cell, or the host cell can be a prokaryotic cell, suchas a bacterial cell. Introduction of the construct into the host cellcan be effected by calcium phosphate transfection, DEAE-Dextran mediatedtransfection, or electroporation. (Davis, L., Dibner, M., Battey, I.,Basic Methods in Molecular Biology, (1986)).

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

Mature proteins can be expressed in mammalian cells, yeast, bacteria, orother cells under the control of appropriate promoters. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the present invention.Appropriate cloning and expression vectors for use with prokaryotic andeukaryotic hosts are described by Sambrook, et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), thedisclosure of which is hereby incorporated by reference.

Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes is increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act on a promoter to increase itstranscription. Examples including the SV40 enhancer on the late side ofthe replication origin bp 100 to 270, a cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), α-factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated protein into the periplasmic space orextracellular medium. Optionally, the heterologous sequence can encode afusion protein including an N-terminal identification peptide impartingdesired characteristics, e.g., stabilization or simplified purificationof expressed recombinant product.

Useful expression vectors for bacterial use are constructed by insertinga structural DNA sequence encoding a desired protein together withsuitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice.

As a representative but nonlimiting example, useful expression vectorsfor bacterial use can comprise a selectable marker and bacterial originof replication derived from commercially available plasmids comprisinggenetic elements of the well known cloning vector pBR322 (ATCC 37017).Such commercial vectors include, for example, pKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wis.,USA). These pBR322 "backbone" sections are combined with an appropriatepromoter and the structural sequence to be expressed.

Following transformation of a suitable host strain and growth of thehost strain to an appropriate cell density, the selected promoter isinduced by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period.

Cells are typically harvested by centrifugation, disrupted by physicalor chemical means, and the resulting crude extract retained for furtherpurification.

Microbial cells employed in expression of proteins can be disrupted byany convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents, such methods arewell know to those skilled in the art.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts, described by Gluzman,Cell, 23:175 (1981), and other cell lines capable of expressing acompatible vector, for example, the C127, 3T3, CHO, HeLa and BHK celllines. Mammalian expression vectors will comprise an origin ofreplication, a suitable promoter and enhancer, and also any necessaryribosome binding sites, polyadenylation site, splice donor and acceptorsites, transcriptional termination sequences, and 5' flankingnontranscribed sequences. DNA sequences derived from the SV40 splice,and polyadenylation sites may be used to provide the requirednontranscribed genetic elements.

The PGF polypeptides can be recovered and purified from recombinant cellcultures by methods including ammonium sulfate or ethanol precipitation,acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. Protein refolding steps can be used, as necessary, incompleting configuration of the mature protein. Finally, highperformance liquid chromatography (HPLC) can be employed for finalpurification steps.

The polypeptides of the present invention may be a naturally purifiedproduct, or a product of chemical synthetic procedures, or produced byrecombinant techniques from a prokaryotic or eukaryotic host (forexample, by bacterial, yeast, higher plant, insect and mammalian cellsin culture). Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. Polypeptides of the inventionmay also include an initial methionine amino acid residue.

PGF polypeptides may be used to reduce or inhibit prostate cancer cellgrowth. PGF may affect a variety of cells in different ways, inducinggrowth in certain cells while inhibiting growth in others. Cancer celllines, including prostatic adenocarcinoma, may be treated with PGF.

PGF may also be employed to promote wound healing, such as first, secondand third degree burns, epidermal and internal incisions and thoseincisions resulting from cosmetic surgery. PGF may also be employed tostimulate tissue regeneration.

The polynucleotides and polypeptides of the present invention may beemployed as research reagents and materials for discovery of treatmentsand diagnostics for human disease.

Fragments of the full length PGF gene may be used as a hybridizationprobe for a cDNA library to isolate the full length PGF gene and toisolate other genes which have a high sequence similarity to the PGFgene or similar biological activity. Probes of this type can be, forexample, between 20 and 2000 base pairs. Preferably, however, the probeshave between 30 and 50 bases. The probe may also be used to identify acDNA clone corresponding to a full length transcript and a genomic cloneor clones that contain the complete PGF gene including regulatory andpromotor regions, exons, and introns. An example of a screen comprisesisolating the coding region of the PGF gene by using the known DNAsequence to synthesize an oligonucleotide probe. Labeledoligonucleotides having a sequence complementary to that of the gene ofthe present invention are used to screen a library of human cDNA,genomic DNA or mRNA to determine which members of the library the probehybridizes to.

This invention provides a method for identification of the receptor forPGF. The gene encoding the receptor can be identified by numerousmethods known to those of skill in the art, for example, ligand panningand FACS sorting (Coligan, et al., Current Protocols in Immun., 1(2),Chapter 5, (1991)). Preferably, expression cloning is employed whereinpolyadenylated RNA is prepared from a cell responsive to PGF, and a cDNAlibrary created from this RNA is divided into pools and used totransfect COS cells or other cells that are not responsive to PGF.Transfected cells which are grown on glass slides are exposed to labeledPGF. PGF can be labeled by a variety of means including iodination orinclusion of a recognition site for a site-specific protein kinase.Following fixation and incubation, the slides are subjected toautoradiographic analysis. Positive pools are identified and sub-poolsare prepared and retransfected using an iterative sub-pooling andrescreening process, eventually yielding a single clone that encodes theputative receptor.

As an alternative approach for receptor identification, labeled PGF canbe photoaffinity linked with cell membrane or extract preparations thatexpress the receptor molecule. Cross-linked material is resolved by PAGEand exposed to X-ray film. The labeled complex containing thePGF-receptor can be excised, resolved into peptide fragments, andsubjected to protein microsequencing. The amino acid sequence obtainedfrom microsequencing would be used to design a set of degenerateoligonucleotide probes to screen a cDNA library to identify the geneencoding the putative receptor.

This invention is also related to a method of screening compounds toidentify those which mimic PGF (agonists) or prevent the effect of PGF.An example of such a method takes advantage of the ability of PGF tosignificantly stimulate the proliferation of human endothelial cells inthe presence of the comitogen Con A. Endothelial cells are obtained andcultured in 96-well flat-bottomed culture plates (Costar, Cambridge,Mass.) in RPM1 1640 supplemented with 10% heat-inactivated fetal bovineserum (Hyclone Labs, Logan, Utah), 1% L-glutamine, 100 U/ml penicillin,100 μg/ml streptomycin, 0.1% gentamicin (Gibco Life Technologies, GrandIsland, N.Y.) in the presence of 2 μg/ml of Con-A (Calbiochem, La Jolla,Calif.). Con-A, and the compound to be screened are added to a finalvolume of 0.2 ml. After 60 h at 37° C., cultures are pulsed with 1 μCiof ³ [H]thymidine (5 Ci/mmol; 1 Ci=37 BGq; NEN) for 12-18 h andharvested onto glass fiber filters (PhD; Cambridge Technology,Watertown, Mass.). Mean ³ [H]thymidine incorporation (cpm) of triplicatecultures is determined using a liquid scintillation counter (BeckmanInstruments, Irvine, Calif.). Significant ³ [H]thymidine incorporationindicates stimulation of endothelial cell proliferation.

Alternatively, the response of a known second messenger system followinginteraction of PGF and receptor would be measured and compared in thepresence or absence of the compound. Such second messenger systemsinclude but are not limited to, CAMP guanylate cyclase, ion channels orphosphoinositide hydrolysis.

To assay for antagonists, the assay described above is performed,however, in this assay PGF is added along with the compound to bescreened and the ability of the compound to inhibit ³ [H]thymidineincorporation in the presence of PGF, indicates that the compound is anantagonist to PGF. Alternatively, PGF antagonists may be detected bycombining PGF and a potential antagonist with membrane-bound PGFreceptors or recombinant receptors under appropriate conditions for acompetitive inhibition assay. PGF can be labeled, such as byradioactivity, such that the number of PGF molecules bound to thereceptor can determine the effectiveness of the potential antagonist.

Alternatively, a mammalian cell or membrane preparation expressing thePGF receptor is incubated with labeled PGF in the presence of thecompound. The ability of the compound to enhance or block thisinteraction could then be measured.

Examples of potential PGF antagonists include an antibody, or in somecases, an oligonucleotide, which binds to the polypeptide.Alternatively, a potential antagonist may be a closely related protein,for example a mutated form of the protein, which binds to the receptorsites, however, they are inactive forms of the polypeptide and therebyprevent the action of PGF since receptor sites are occupied.

Another potential PGF antagonist is an antisense construct preparedusing antisense technology. Antisense technology can be used to controlgene expression through triple-helix formation or antisense DNA or RNA,both of which methods are based on binding of a polynucleotide to DNA orRNA. For example, the 5' coding portion of the polynucleotide sequence,which encodes for the mature polypeptides of the present invention, isused to design an antisense RNA oligonucleotide of from about 10 to 40base pairs in length. A DNA oligonucleotide is designed to becomplementary to a region of the gene involved in transcription (triplehelix--see Lee et al., Nucl. Acids Res., 6:3073 (1979); Cooney et al,Science, 241:456 (1988); and Dervan et al., Science, 251: 1360 (1991)),thereby preventing transcription and the production of PGF. Theantisense RNA oligonucleotide hybridizes to the mRNA in vivo and blockstranslation of the mRNA molecule into PGF polypeptide (Antisense-Okano,J. Neurochem., 56:560 (1991); Oligodeoxynucleotides as AntisenseInhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)). Theoligonucleotides described above can also be delivered to cells suchthat the antisense RNA or DNA may be expressed in vivo to inhibitproduction of PGF.

Potential PGF antagonists include a small molecule which binds to andoccupies the active site of the polypeptide thereby making itinaccessible to substrate such that normal biological activity isprevented. Examples of small molecules include but are not limited tosmall peptides or peptide-like molecules and non-peptide molecules.

The antagonists may be employed to treat PGF-dependent prostate cancerand benign prostatic hyperplasia (BPH). The antagonists may be employedin a composition with a pharmaceutically acceptable carrier, e.g., ashereinafter described.

The PGF polypeptides and agonists and antagonists may be employed incombination with a suitable pharmaceutical carrier. Such compositionscomprise a therapeutically effective amount of the polypeptide, and apharmaceutically acceptable carrier or excipient. Such a carrierincludes but is not limited to saline, buffered saline, dextrose, water,glycerol, ethanol, and combinations thereof. The formulation should suitthe mode of administration.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration. In addition, thepharmaceutical compositions may be employed in conjunction with othertherapeutic compounds.

The pharmaceutical compositions may be administered in a convenientmanner such as by the oral, topical, intravenous, intraperitoneal,intramuscular, subcutaneous, intranasal or intradermal routes. Thepharmaceutical compositions are administered in an amount which iseffective for treating and/or prophylaxis of the specific indication. Ingeneral, they are administered in an amount of at least about 10 μg/kgbody weight and in most cases they will be administered in an amount notin excess of about 8 mg/Kg body weight per day. In most cases, thedosage is from about 10 μg/kg to about 1 mg/kg body weight daily, takinginto account the routes of administration, symptoms, etc.

The PGF polypeptides, and agonists and antagonists which arepolypeptides, may also be employed in accordance with the presentinvention by expression of such polypeptides in vivo, which is oftenreferred to as "gene therapy."

Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with theengineered cells then being provided to a patient to be treated with thepolypeptide. Such methods are well-known in the art. For example, cellsmay be engineered by procedures known in the art by use of a retroviralparticle containing RNA encoding a polypeptide of the present invention.

Similarly, cells may be engineered in vivo for expression of apolypeptide in vivo by, for example, procedures known in the art. Asknown in the art, a producer cell for producing a retroviral particlecontaining RNA encoding the polypeptide of the present invention may beadministered to a patient for engineering cells in vivo and expressionof the polypeptide in vivo. These and other methods for administering apolypeptide of the present invention by such method should be apparentto those skilled in the art from the teachings of the present invention.For example, the expression vehicle for engineering cells may be otherthan a retrovirus, for example, an adenovirus which may be used toengineer cells in vivo after combination with a suitable deliveryvehicle.

This invention is also related to the use of the PGF gene as adiagnostic. Detection of a mutated form of PGF will allow a diagnosis ofa disease or a susceptibility to a disease which results fromunderexpression of PGF.

Individuals carrying mutations in the human PGF gene may be detected atthe DNA level by a variety of techniques. Nucleic acids for diagnosismay be obtained from a patient's cells, such as from blood, urine,saliva, tissue biopsy and autopsy material. The genomic DNA may be useddirectly for detection or may be amplified enzymatically by using PCR(Saiki et al., Nature, 324:163-166 (1986)) prior to analysis. RNA orcDNA may also be used for the same purpose. As an example, PCR primerscomplementary to the nucleic acid encoding PGF can be used to identifyand analyze PGF mutations. For example, deletions and insertions can bedetected by a change in size of the amplified product in comparison tothe normal genotype. Point mutations can be identified by hybridizingamplified DNA to radiolabeled PGF RNA or alternatively, radiolabeled PGFantisense DNA sequences. Perfectly matched sequences can bedistinguished from mismatched duplexes by RNase A digestion or bydifferences in melting temperatures.

Genetic testing based on DNA sequence differences may be achieved bydetection of alteration in electrophoretic mobility of DNA fragments ingels with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamide gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures (see, e.g., Myerset al., Science, 230:1242 (1985)).

Sequence changes at specific locations may also be revealed by nucleaseprotection assays, such as RNase and S1 protection or the chemicalcleavage method (e.g., Cotton et al., PNAS, USA, 85:4397-4401 (1985)).

Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing or the use of restriction enzymes, (e.g.,Restriction Fragment Length Polymorphisms (RFLP)) and Southern blottingof genomic DNA.

In addition to more conventional gel-electrophoresis and DNA sequencing,mutations can also be detected by in situ analysis.

The present invention also relates to a diagnostic assay for detectingaltered levels of PGF protein in various tissues since anover-expression of the proteins compared to normal control tissuesamples allows early detection of prostate cancer or benign prostatichyperplasia. Assays used to detect levels of PGF protein in a samplederived from a host are well-known to those of skill in the art andinclude radioimmunoassays, competitive-binding assays, Western Blotanalysis and preferably an ELISA assay.

An Elisa assay initially comprises preparing an antibody specific to thePGF antigen, preferably a monoclonal antibody. In addition a reporterantibody is prepared against the monoclonal antibody. To the reporterantibody is attached a detectable reagent such as radioactivity,fluorescence or in this example a horseradish peroxidase enzyme. Asample is now removed from a host and incubated on a solid support, e.g.a polystyrene dish, that binds the proteins in the sample. Any freeprotein binding sites on the dish are then covered by incubating with anon-specific protein like BSA. Next, the monoclonal antibody isincubated in the dish during which time the monoclonal antibodies attachto any PGF proteins attached to the polystyrene dish. All unboundmonoclonal antibody is washed out with buffer. The reporter antibodylinked to horseradish peroxidase is now placed in the dish resulting inbinding of the reporter antibody to any monoclonal antibody bound toPGF. Unattached reporter antibody is then washed out. Peroxidasesubstrates are then added to the dish and the amount of color developedin a given time period is a measurement of the amount of PGF proteinpresent in a given volume of patient sample when compared against astandard curve.

A competition assay may be employed wherein antibodies specific to PGFis attached to a solid support and labeled PGF and a sample derived fromthe host are passed over the solid support and the amount of labeldetected attached to the solid support can be correlated to a quantityof PGF in the sample.

The sequences of the present invention are also valuable for chromosomeidentification. The sequence is specifically targeted to and canhybridize with a particular location on an individual human chromosome.Moreover, there is a current need for identifying particular sites onthe chromosome. Few chromosome marking reagents based on actual sequencedata (repeat polymorphisms) are presently available for markingchromosomal location. The mapping of DNAs to chromosomes according tothe present invention is an important first step in correlating thosesequences with genes associated with disease.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers(preferably 15-25 bp) from the cDNA. Computer analysis of the 3'untranslated region of the gene is used to rapidly select primers thatdo not span more than one exon in the genomic DNA, thus complicating theamplification process. These primers are then used for PCR screening ofsomatic cell hybrids containing individual human chromosomes. Only thosehybrids containing the human gene corresponding to the primer will yieldan amplified fragment.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular DNA to a particular chromosome. Using the present inventionwith the same oligonucleotide primers, sublocalization can be achievedwith panels of fragments from specific chromosomes or pools of largegenomic clones in an analogous manner. Other mapping strategies that cansimilarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific cDNAlibraries.

Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphasechromosomal spread can be used to provide a precise chromosomal locationin one step. This technique can be used with cDNA as short as 500 or 600bases; however, clones larger than 2,000 bp have a higher likelihood ofbinding to a unique chromosomal location with sufficient signalintensity for simple detection. FISH requires use of the clones fromwhich the express sequence tag (EST) was derived, and the longer thebetter. For example, 2,000 bp is good, 4,000 is better, and more than4,000 is probably not necessary to get good results a reasonablepercentage of the time. For a review of this technique, see Verma etal., Human Chromosomes: a Manual of Basic Techniques, Pergamon Press,New York (1988).

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. Such data are found, for example, in V. McKusick,Mendelian Inheritance in Man (available on line through Johns HopkinsUniversity Welch Medical Library). The relationship between genes anddiseases that have been mapped to the same chromosomal region are thenidentified through linkage analysis (coinheritance of physicallyadjacent genes).

Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

The polypeptides, their fragments or other derivatives, or analogsthereof, or cells expressing them can be used as an immunogen to produceantibodies thereto. These antibodies can be, for example, polyclonal ormonoclonal antibodies. The present invention also includes chimeric,single chain, and humanized antibodies, as well as Fab fragments, or theproduct of an Fab expression library. Various procedures known in theart may be used for the production of such antibodies and fragments.

Antibodies generated against the polypeptides corresponding to asequence of the present invention can be obtained by direct injection ofthe polypeptides into an animal or by administering the polypeptides toan animal, preferably a nonhuman. The antibody so obtained will thenbind the polypeptides itself. In this manner, even a sequence encodingonly a fragment of the polypeptides can be used to generate antibodiesbinding the whole native polypeptides. Such antibodies can then be usedto isolate the polypeptide from tissue expressing that polypeptide.

For preparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique (Kohler and Milstein, 1975,Nature, 256:495-497), the trioma technique, the human B-cell hybridomatechnique (Kozbor et al., 1983, Immunology Today 4:72), and theEBV-hybridoma technique to produce human monoclonal antibodies (Cole, etal., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc., pp. 77-96).

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies toimmunogenic polypeptide products of this invention. Also, transgenicmice may be used to express humanized antibodies to immunogenicpolypeptide products of this invention.

The present invention will be further described with reference to thefollowing examples; however, it is to be understood that the presentinvention is not limited to such examples. All parts or amounts, unlessotherwise specified, are by weight.

In order to facilitate understanding of the following examples certainfrequently occurring methods and/or terms will be described.

"Plasmids" are designated by a lower case p preceded and/or followed bycapital letters and/or numbers. The starting plasmids herein are eithercommercially available, publicly available on an unrestricted basis, orcan be constructed from available plasmids in accord with publishedprocedures. In addition, equivalent plasmids to those described areknown in the art and will be apparent to the ordinarily skilled artisan.

"Digestion" of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 μg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37° C. are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion the reaction is electrophoreseddirectly on a polyacrylamide gel to isolate the desired fragment.

Size separation of the cleaved fragments is performed using 8 percentpolyacrylamide gel described by Goeddel, D. et al., Nucleic Acids Res.,8:4057 (1980).

"Oligonucleotides" refers to either a single strandedpolydeoxynucleotide or two complementary polydeoxynucleotide strandswhich may be chemically synthesized. Such synthetic oligonucleotideshave no 5' phosphate and thus will not ligate to another oligonucleotidewithout adding a phosphate with an ATP in the presence of a kinase. Asynthetic oligonucleotide will ligate to a fragment that has not beendephosphorylated.

"Ligation" refers to the process of forming phosphodiester bonds betweentwo double stranded nucleic acid fragments (Maniatis, T., et al., Id.,p. 146). Unless otherwise provided, ligation may be accomplished usingknown buffers and conditions with 10 units of T4 DNA ligase ("ligase")per 0.5 μg of approximately equimolar amounts of the DNA fragments to beligated.

Unless otherwise stated, transformation was performed as described inthe method of Graham, F. and Van der Eb, A., Virology, 52:456-457(1973).

EXAMPLE 1 Bacterial Expression and Purification of PGF

The DNA sequence encoding PGF, ATCC #75902, is initially amplified usingPCR oligonucleotide primers corresponding to the 5' and 3' termini.Additional nucleotides corresponding to PGF are added to the 5 ' and 3'sequences, respectively. The 5' oligonucleotide primer has the sequence5' CGCGCGAAGCTTATGCTCCTGGTGTTGCTGGTG 3' (SEQ ID NO:5) contains a HindIIIrestriction enzyme site followed by 21 nucleotides of PGF codingsequence starting from the first amino acid. The 3' sequence 5'GCGCGCTCTAGATCATATGCAGTGGCAGTCTTT 3' (SEQ ID NO:6) containscomplementary sequences to an XbaI site and is followed by 21nucleotides of PGF. The restriction enzyme sites correspond to therestriction enzyme sites on the bacterial expression vector pQE-9(Qiagen, Inc. 9259 Eton Avenue, Chatsworth, Calif., 91311). pQE-9encodes antibiotic resistance (Amp^(r)), a bacterial origin ofreplication (ori), an IPTG-regulatable promoter operator (P/O), aribosome binding site (RBS), a 6-His tag and restriction enzyme sites.pQE-9 is then digested with BamHI and XbaI. The amplified sequences areligated into pQE-9 and are inserted in frame with the sequence encodingfor the histidine tag and the RBS. The ligation mixture is then used totransform E. coli strain m15/pREP4 (Qiagen) by the procedure describedin Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, ColdSpring Laboratory Press, (1989). M15/pREP4 contains multiple copies ofthe plasmid pREP4, which expresses the lacI repressor and also conferskanamycin resistance (Kan^(r)). Transformants are identified by theirability to grow on LB plates and ampicillin/kanamycin resistant coloniesare selected. Plasmid DNA is isolated and confirmed by restrictionanalysis. Clones containing the desired constructs are grown overnight(O/N) in liquid culture in LB media supplemented with both Amp (100ug/ml) and Kan (25 ug/ml). The O/N culture is used to inoculate a largeculture at a ratio of 1:100 to 1:250. The cells are grown to an opticaldensity 600 (O.D.⁶⁰⁰) of between 0.4 and 0.6. IPTG("Isopropyl-B-D-thiogalacto pyranoside") is then added to a finalconcentration of 1 mM. IPTG induces by inactivating the lacI repressor,clearing the P/O leading to increased gene expression. Cells are grownan extra 3 to 4 hours. Cells are then harvested by centrifugation. Thecell pellet is solubilized in the chaotropic agent 6 Molar GuanidineHCl. After clarification, solubilized PGF is purified from this solutionby chromatography on a Nickel-Chelate column under conditions that allowfor tight binding by proteins containing the 6-His tag (Hochuli, E. etal., J. Chromatography 411:177-184 (1984)). PGF is eluted from thecolumn in 6 molar guanidine HCl pH 5.0 and for the purpose ofrenaturation adjusted to 3 molar guanidine HCl, 100 mM sodium phosphate,10 mmolar glutathione (reduced) and 2 mmolar glutathione (oxidized).After incubation in this solution for 12 hours the protein is dialyzedto 10 mmolar sodium phosphate.

EXAMPLE 2 Expression of Recombinant PGF in COS cells

The expression of plasmid, PGF HA is derived from a vector N11containing: 1) SV40 origin of replication, 2) ampicillin resistancegene, 3) E. coli replication origin, 4) CMV promoter followed by apolylinker region, a rat preproinsulin 3' intron and polyadenylationsite. A DNA fragment encoding the entire PGF precursor and a HA tagfused in frame to its 3' end was cloned into the polylinker region ofthe vector, therefore, the recombinant protein expression is directedunder the CMV promoter. The HA tag correspond to an epitope derived fromthe influenza hemagglutinin protein as previously described (I. Wilson,H. Niman, R. Heighten, A Cherenson, M. Connolly, and R. Lerner, 1984,Cell 37, 767). The infusion of HA tag to our target protein allows easydetection of the recombinant protein with an antibody that recognizesthe HA epitope.

The plasmid construction strategy is described as follows:

The DNA sequence encoding PGF, ATCC #75902, was constructed by PCR usingtwo primers: the 5' primer 5' GCGCAGATCTGCCACCATGCTCCTGGTGTTGCTGGTGCTG3' (SEQ ID NO:7) contains a Bgl II site followed by 24 nucleotides ofPGF coding sequence starting from the initiation codon; the 3' sequence5' CGCGAGATCTTCAAGCGTAGTCTGGGACGTCGTATGGGTATATGCAGTGGCAGTCTTTGGC 3' (SEQID NO:8) contains complementary sequences to a Bgl II site, translationstop codon, HA tag and the last 21 nucleotides of the PGF codingsequence (not including the stop codon). Therefore, the PCR productcontains a Bgl II site, PGF coding sequence followed by HA tag fused inframe, a translation termination stop codon next to the HA tag, and anBgl II site. The PCR amplified DNA fragment and the vector, N11 weredigested with Bgl II restriction enzyme and ligated. The ligationmixture was transformed into E. coli strain DH5α (Stratagene CloningSystems, La Jolla, Calif.) the transformed culture was plated onampicillin media plates and resistant colonies were selected. PlasmidDNA was isolated from transformants and examined by restriction analysisfor the presence of the correct fragment. For expression of therecombinant PGF, COS cells were transfected with the expression vectorby DEAE-DEXTRAN method (J. Sambrook, E. Fritsch, T. Maniatis, MolecularCloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989)). Theexpression of the PGF HA protein was detected by radiolabelling andimmunoprecipitation method (E. Harlow, D. Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, (1988)). Cells werelabelled for 8 hours with ³⁵ S-cysteine two days post transfection.Culture media were then collected and cells were lysed with detergent(RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mMTris, pH 7.5) (Wilson, I. et al., Id. 37:767 (1984)). Both cell lysateand culture media were precipitated with a HA specific monoclonalantibody. Proteins precipitated were analyzed on 15% SDS-PAGE gels.

EXAMPLE 3 Expression Pattern of PGF in Human Tissue

Northern blot analysis was carried out to examine the levels ofexpression of PGF in human tissues. Total cellular RNA samples wereisolated with RNAzol™ B system (Biotecx Laboratories, Inc. 6023 SouthLoop East, Houston, Tex. 77033). About 10 μg of total RNA isolated fromeach human tissue specified was separated on 1% agarose gel and blottedonto a nylon filter (Sambrook, Fritsch, and Maniatis, Molecular Cloning,Cold Spring Harbor Press, (1989)). The labeling reaction was doneaccording to the Stratagene Prime-It kit with 50 ng DNA fragment. Thelabeled DNA was purified with a Select-G-50 column (5 Prime-3 Prime,Inc. Boulder, Colo. 80303). The filter was then hybridized withradioactive labeled full length PGF gene at 1,000,000 cpm/ml in 0.5 MNaPO₄, pH 7.4 and 7% SDS overnight at 65° C. After wash twice at roomtemperature and twice at 60° C. with 0.5×SSC, 0.1% SDS, the filter wasthen exposed at -70° C. overnight with an intensifying screen. Themessage RNA for PGF is abundant in the prostate.

EXAMPLE 4 Expression of Recombinant PGF in CHO Cells

The vector pN346 is used for the expression of the PGF protein. PlasmidpN346 is a derivative of the plasmid pSV2-dhfr [ATCC Accession No.37146]. Both plasmids contain the mouse dhfr gene under control of theSV40 early promoter. Chinese hamster ovary--or other cells lackingdihydrofolate activity that are transfected with these plasmids can beselected by growing the cells in a selective medium (alpha minus MEM,Lift Technologies) supplemented with the chemotherapeutic agentmethotrexate. The amplification of the DHFR genes in cells resistant tomethotrexate (MTX) has been well documented (see, e.g., Alt, F. W.,Kellems, R. M., Bertino, J. R., and Schimke, R. T., 1978, J. Biol. Chem.253:1357-1370, Hamlin, J. L. and Ma, C. 1990, Biochem. et Biophys. Acta,1097:107-143, Page, M. J. and Sydenham, M. A. 1991, Biotechnology Vol.9:64-68). Cells grown in increasing concentrations of MTX developresistance to the drug by overproducing the target enzyme, DHFR, as aresult of amplification of the DHFR gene. If a second gene is linked tothe dhfr gene it is usually co-amplified and over-expressed. It is stateof the art to develop cell lines carrying more than 1,000 copies of thegenes. Subsequently, when the methotrexate is withdrawn, cell linescontain the amplified gene integrated into the chromosome(s).

Plasmid pN346 contains for the expression of the gene of interest astrong promoter of the long terminal repeat (LTR) of the Rouse SarcomaVirus (Cullen, et al., Molecular and Cellular Biology, March 1985,438-447) plus a fragment isolated from the enhancer of the immediateearly gene of human cytomegalovirus (C4V) (Boshart et al., Cell41:521-530, 1985). Downstream of the promoter are the following singlerestriction enzyme cleavage sites that allow the integration of thegenes: BamHI, Pvull, and Nrul. Behind these cloning sites the plasmidcontains translational stop codons in all three reading frames followedby the 3' intron and the polyadenylation site of the rat preproinsulingene. Other high efficient promoters can also be used for theexpression, e.g., the human β-actin promoter, the SV40 early or latepromoters or the long terminal repeats from other retroviruses, e.g.,HIV and HTLVI. For the polyadenylation of the mRNA other signals, e.g.,from the human growth hormone or globin genes can be used as well.

Stable cell lines carrying a gene of interest integrated into thechromosome can also be selected upon co-transfection with a selectablemarker such as gpt, G418 or hygromycin. It is advantageous to use morethan one selectable marker in the beginning, e.g. G418 plusmethotrexate.

The plasmid pN346 was digested with the restriction enzyme BamHI andthen dephosphorylated using calf intestinal phosphatase by proceduresknown in the art. The vector was then isolated from a 1% agarose gel.

The DNA sequence encoding PGF, ATCC #75902, was amplified using PCRoligonucleotide primers corresponding to the 5' and 3' sequences of thegene:

The 5 ' primer has the sequence GCGCAGATCTGCCACCATGCTCCTGGTGTTGCT (SEQID NO:9) and contains a BglII restriction enzyme site (in bold) followedby 17 nucleotides resembling an efficient signal for translation (Kozak,M., supra) plus the first 17 nucleotides of the gene (the initiationcodon for translation "ATG" is underlined.).

The 3' primer has the sequence 5' CGCGAGATCTTCATATGCAGTGGCAGTCTTTGGC 3'(SEQ ID NO:10) and contains the cleavage site for the restrictionendonuclease BglII (in bold) and 20 nucleotides complementary to the 3'non-translated sequence of the gene.

The amplified fragments were isolated from a 1% agarose gel as describedabove and then digested with the endonuclease BglII and then purifiedagain on a 1% agarose gel.

The isolated fragment and the dephosphorylated vector were then ligatedwith T4 DNA ligase. E. coli HB101 cells were then transformed andbacteria identified that contained the plasmid pN346 inserted in thecorrect orientation using the restriction enzymes BamHI. The sequence ofthe inserted gene was confirmed by DNA sequencing.

Transfection of CHO-dhfr-cells

Chinese hamster ovary cells lacking an active DHFR enzyme were used fortransfection. 5 μg of the expression plasmid N346 were cotransfectedwith 0.5 μg of the plasmid pSvneo using the lipofectin method (Felgneret al., supra). The plasmid pSV2-neo contains a dominant selectablemarker, the gene neo from Tn5 encoding an enzyme that confers resistanceto a group of antibiotics including G418. The cells were seeded in alphaminus MEM supplemented with 1 mg/ml G418. After 2 days, the cells weretrypsinized and seeded in hybridoma cloning plates (Greiner, Germany)and cultivated from 10-14 days. After this period, single clones weretrypsinized and then seeded in 6-well petri dishes using differentconcentrations of methotrexate (25, 50 nm, 100 nm, 200 nm, 400 nm).Clones growing at the highest concentrations of methotrexate were thentransferred to new 6-well plates containing even higher concentrationsof methotrexate (500 nM, 1 μM, 2 μM, 5 μM). The same procedure wasrepeated until clones grew at a concentration of 100 μM.

The expression of the desired gene product was analyzed by Western blotanalysis and SDS-PAGE.

Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, within thescope of the appended claims, the invention may be practiced otherwisethan as particularly described.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                  - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 10                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1024 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 64..948                                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - GCACACGAGG CAACCTGCAC AGCCATGCCC GGGCAAGAAC TCAGGACGCT GA -             #ATGGCTCT     60                                                                 - - CAG ATG CTC CTG GTG TTG CTG GTG CTC TCG TG - #G CTG CCG CAT GGG        GGC      108                                                                        Met Leu Leu Val Leu Leu Val Leu - #Ser Trp Leu Pro His Gly Gly                 1            - #   5               - #   10               - #   15       - - GCC CTG TCT CTG GCC GAG GCG AGC CGC GCA AG - #T TTC CCG GGA CCC TCA          156                                                                       Ala Leu Ser Leu Ala Glu Ala Ser Arg Ala Se - #r Phe Pro Gly Pro Ser                            20 - #                 25 - #                 30              - - GAG TTG CAC TCC GAA GAC TCC AGA TTC CGA GA - #G TTG CGG AAA CGC TAC          204                                                                       Glu Leu His Ser Glu Asp Ser Arg Phe Arg Gl - #u Leu Arg Lys Arg Tyr                        35     - #             40     - #             45                  - - GAG GAC CTG CTA ACC AGG CTG CGG GCC AAC CA - #G AGC TGG GAA GAT TCG          252                                                                       Glu Asp Leu Leu Thr Arg Leu Arg Ala Asn Gl - #n Ser Trp Glu Asp Ser                    50         - #         55         - #         60                      - - AAC ACC GAC CTC GTC CCG GCC CCT GCA GTC CG - #G ATA CTC ACG CCA GAA          300                                                                       Asn Thr Asp Leu Val Pro Ala Pro Ala Val Ar - #g Ile Leu Thr Pro Glu                65             - #     70             - #     75                          - - GTG CGG CTG GGA TCC GGC GGC CAC CTG CAC CT - #G CGT ATC TCT CGG GCC          348                                                                       Val Arg Leu Gly Ser Gly Gly His Leu His Le - #u Arg Ile Ser Arg Ala            80                 - # 85                 - # 90                 - # 95       - - GCC CTT CCC GAG GGG CTC CCC GAG GCC TCC CG - #C CTT CAC CGG GCT CTG          396                                                                       Ala Leu Pro Glu Gly Leu Pro Glu Ala Ser Ar - #g Leu His Arg Ala Leu                           100  - #               105  - #               110              - - TTC CGG CTG TCC CCG ACG GCG TCA AGG TCG TG - #G GAC GTG ACA CGA CCG          444                                                                       Phe Arg Leu Ser Pro Thr Ala Ser Arg Ser Tr - #p Asp Val Thr Arg Pro                       115      - #           120      - #           125                  - - CTG CGG CGT CAG CTC AGC CTT GCA AGA CCC CA - #G GCG CCC GCG CTG CAC          492                                                                       Leu Arg Arg Gln Leu Ser Leu Ala Arg Pro Gl - #n Ala Pro Ala Leu His                   130          - #       135          - #       140                      - - CTG CGA CTG TCG CCG CCG CCG TCG CAG TCG GA - #C CAA CTG CTG GCA GAA          540                                                                       Leu Arg Leu Ser Pro Pro Pro Ser Gln Ser As - #p Gln Leu Leu Ala Glu               145              - #   150              - #   155                          - - TCT TCG TCC GCA CGG CCC CAG CTG GAG TTG CA - #C TTG CGG CCG CAA GCC          588                                                                       Ser Ser Ser Ala Arg Pro Gln Leu Glu Leu Hi - #s Leu Arg Pro Gln Ala           160                 1 - #65                 1 - #70                 1 -      #75                                                                              - - GCC AGG GGG CGC CGC AGA GCG CGT GCG CGC AA - #C GGG GAC CAC TGT        CCG      636                                                                    Ala Arg Gly Arg Arg Arg Ala Arg Ala Arg As - #n Gly Asp His Cys Pro                          180  - #               185  - #               190              - - CTC GGG CCC GGG CGT TGC TGC CGT CTG CAC AC - #G GTC CGC GCG TCG CTG          684                                                                       Leu Gly Pro Gly Arg Cys Cys Arg Leu His Th - #r Val Arg Ala Ser Leu                       195      - #           200      - #           205                  - - GAA GAC CTG GGC TGG GCC GAT TGG GTG CTG TC - #G CCA CGG GAG GTG CAA          732                                                                       Glu Asp Leu Gly Trp Ala Asp Trp Val Leu Se - #r Pro Arg Glu Val Gln                   210          - #       215          - #       220                      - - GTG ACC ATG TGC ATC GGC GCG TGC CCG AGC CA - #G TTC CGG GCG GCA AAC          780                                                                       Val Thr Met Cys Ile Gly Ala Cys Pro Ser Gl - #n Phe Arg Ala Ala Asn               225              - #   230              - #   235                          - - ATG CAC GCG CAG ATC AAG ACG AGC CTG CAC CG - #C CTG AAG CCC GAC ACG          828                                                                       Met His Ala Gln Ile Lys Thr Ser Leu His Ar - #g Leu Lys Pro Asp Thr           240                 2 - #45                 2 - #50                 2 -      #55                                                                              - - GTG CCA GCG CCC TGC TGC GTG CCC GCC AGC TA - #C AAT CCC ATG GTG        CTC      876                                                                    Val Pro Ala Pro Cys Cys Val Pro Ala Ser Ty - #r Asn Pro Met Val Leu                          260  - #               265  - #               270              - - ATT CAA AAG ACC GAC ACC GGG GTG TCG TTC CA - #A ACC TAT GAT GAC TTG          924                                                                       Ile Gln Lys Thr Asp Thr Gly Val Ser Phe Gl - #n Thr Tyr Asp Asp Leu                       275      - #           280      - #           285                  - - TTA GCC AAA GAC TGC CAC TGC ATA TGAGCAGTCC TG - #GTCCTTCC ACTGTGCACC         978                                                                       Leu Ala Lys Asp Cys His Cys Ile                                                       290          - #       295                                             - - TGCGCGGGGG ACGGGACCTC AGTTGTCCTG CCCTGTGGAA TGCGCT   - #                   1024                                                                         - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 295 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - Met Leu Leu Val Leu Leu Val Leu Ser Trp Le - #u Pro His Gly Gly Ala        1               5 - #                 10 - #                 15              - - Leu Ser Leu Ala Glu Ala Ser Arg Ala Ser Ph - #e Pro Gly Pro Ser Glu                   20     - #             25     - #             30                  - - Leu His Ser Glu Asp Ser Arg Phe Arg Glu Le - #u Arg Lys Arg Tyr Glu               35         - #         40         - #         45                      - - Asp Leu Leu Thr Arg Leu Arg Ala Asn Gln Se - #r Trp Glu Asp Ser Asn           50             - #     55             - #     60                          - - Thr Asp Leu Val Pro Ala Pro Ala Val Arg Il - #e Leu Thr Pro Glu Val       65                 - # 70                 - # 75                 - # 80       - - Arg Leu Gly Ser Gly Gly His Leu His Leu Ar - #g Ile Ser Arg Ala Ala                       85 - #                 90 - #                 95              - - Leu Pro Glu Gly Leu Pro Glu Ala Ser Arg Le - #u His Arg Ala Leu Phe                  100      - #           105      - #           110                  - - Arg Leu Ser Pro Thr Ala Ser Arg Ser Trp As - #p Val Thr Arg Pro Leu              115          - #       120          - #       125                      - - Arg Arg Gln Leu Ser Leu Ala Arg Pro Gln Al - #a Pro Ala Leu His Leu          130              - #   135              - #   140                          - - Arg Leu Ser Pro Pro Pro Ser Gln Ser Asp Gl - #n Leu Leu Ala Glu Ser      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Ser Ser Ala Arg Pro Gln Leu Glu Leu His Le - #u Arg Pro Gln Ala        Ala                                                                                             165  - #               170  - #               175             - - Arg Gly Arg Arg Arg Ala Arg Ala Arg Asn Gl - #y Asp His Cys Pro Leu                  180      - #           185      - #           190                  - - Gly Pro Gly Arg Cys Cys Arg Leu His Thr Va - #l Arg Ala Ser Leu Glu              195          - #       200          - #       205                      - - Asp Leu Gly Trp Ala Asp Trp Val Leu Ser Pr - #o Arg Glu Val Gln Val          210              - #   215              - #   220                          - - Thr Met Cys Ile Gly Ala Cys Pro Ser Gln Ph - #e Arg Ala Ala Asn Met      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - His Ala Gln Ile Lys Thr Ser Leu His Arg Le - #u Lys Pro Asp Thr        Val                                                                                             245  - #               250  - #               255             - - Pro Ala Pro Cys Cys Val Pro Ala Ser Tyr As - #n Pro Met Val Leu Ile                  260      - #           265      - #           270                  - - Gln Lys Thr Asp Thr Gly Val Ser Phe Gln Th - #r Tyr Asp Asp Leu Leu              275          - #       280          - #       285                      - - Ala Lys Asp Cys His Cys Ile                                                  290              - #   295                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 432 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - Met His Val Arg Ser Leu Arg Ala Ala Ala Pr - #o His Ser Phe Val Ala      1               5   - #                10  - #                15               - - Leu Trp Ala Pro Leu Phe Leu Leu Arg Ser Al - #a Leu Ala Asp Phe Ser                  20      - #            25      - #            30                   - - Leu Asp Asn Glu Val His Ser Ser Phe Ile Hi - #s Arg Arg Leu Arg Ser              35          - #        40          - #        45                       - - Gln Glu Arg Arg Glu Met Gln Arg Glu Ile Le - #u Ser Ile Leu Gly Leu          50              - #    55              - #    60                           - - Pro His Arg Pro Arg Pro His Leu Gln Gly Ly - #s His Asn Ser Ala Pro      65                  - #70                  - #75                  - #80        - - Met Phe Met Leu Asp Leu Tyr Asn Ala Met Al - #a Val Glu Glu Gly Gly                      85  - #                90  - #                95               - - Gly Pro Gly Gly Gln Gly Phe Ser Tyr Pro Ty - #r Lys Ala Val Phe Ser                  100      - #           105      - #           110                  - - Thr Gln Gly Pro Pro Leu Ala Ser Leu Gln As - #p Glu Ser His Phe Leu              115          - #       120          - #       125                      - - Thr Asp Ala Asp Met Val Met Ser Phe Val As - #n Leu Val Glu His Asp          130              - #   135              - #   140                          - - Lys Glu Phe Phe His Pro Arg Tyr His His Ar - #g Glu Phe Arg Phe Asp      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Leu Ser Lys Ile Pro Glu Gly Glu Ala Val Th - #r Ala Ala Glu Phe        Arg                                                                                             165  - #               170  - #               175             - - Ile Tyr Lys Asp Tyr Ile Arg Glu Arg Phe As - #p Asn Glu Thr Phe Arg                  180      - #           185      - #           190                  - - Ile Ser Val Tyr Gln Val Leu Gln Glu His Le - #u Gly Arg Glu Ser Asp              195          - #       200          - #       205                      - - Leu Phe Leu Leu Asp Ser Arg Thr Leu Trp Al - #a Ser Glu Glu Gly Trp          210              - #   215              - #   220                          - - Leu Val Phe Asp Ile Thr Ala Thr Ser Asn Hi - #s Trp Val Val Asn Pro      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Arg His Asn Leu Gly Leu Gln Leu Ser Val Gl - #u Thr Leu Asp Gly        Gln                                                                                             245  - #               250  - #               255             - - Ser Ile Asn Pro Lys Leu Ala Gly Leu Ile Gl - #y Arg His Gly Pro Gln                  260      - #           265      - #           270                  - - Asn Lys Gln Pro Phe Met Val Ala Phe Phe Ly - #s Ala Thr Glu Val His              275          - #       280          - #       285                      - - Phe Arg Ser Ile Arg Ser Thr Gly Ser Lys Gl - #n Arg Ser Gln Asn Arg          290              - #   295              - #   300                          - - Ser Lys Thr Pro Lys Asn Gln Glu Ala Leu Ar - #g Met Ala Asn Val Ala      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Glu Asn Ser Ser Ser Asp Gln Arg Gln Ala Cy - #s Lys Lys His Glu        Leu                                                                                             325  - #               330  - #               335             - - Tyr Val Ser Phe Arg Asp Leu Gly Trp Gln As - #p Trp Ile Ile Ala Pro                  340      - #           345      - #           350                  - - Glu Gly Tyr Ala Ala Tyr Tyr Cys Glu Gly Gl - #u Cys Ala Phe Pro Leu              355          - #       360          - #       365                      - - Asn Ser Tyr Met Asn Ala Thr Asn His Ala Il - #e Val Gln Thr Leu Val          370              - #   375              - #   380                          - - His Phe Ile Asn Pro Glu Thr Val Pro Lys Pr - #o Cys Cys Ala Pro Thr      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Gln Leu Asn Ala Ile Ser Val Leu Tyr Phe As - #p Asp Ser Ser Asn        Val                                                                                             405  - #               410  - #               415             - - Ile Leu Lys Lys Tyr Arg Asn Met Val Val Ar - #g Ala Cys Gly Cys His                  420      - #           425      - #           430                  - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 360 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - Met Val Trp Leu Arg Leu Trp Ala Phe Leu Hi - #s Ile Leu Ala Ile Val      1               5   - #                10  - #                15               - - Thr Leu Asp Pro Glu Leu Lys Arg Arg Glu Gl - #u Leu Phe Leu Arg Ser                  20      - #            25      - #            30                   - - Leu Gly Phe Ser Ser Lys Pro Asn Pro Val Se - #r Pro Pro Pro Val Pro              35          - #        40          - #        45                       - - Ser Ile Leu Trp Arg Ile Phe Asn Gln Arg Me - #t Gly Ser Ser Ile Gln          50              - #    55              - #    60                           - - Lys Lys Lys Pro Asp Leu Cys Phe Val Glu Gl - #u Phe Asn Val Pro Gly      65                  - #70                  - #75                  - #80        - - Ser Val Ile Arg Val Phe Pro Asp Gln Gly Ar - #g Phe Ile Ile Pro Tyr                      85  - #                90  - #                95               - - Ser Asp Asp Ile His Pro Thr Gln Cys Leu Gl - #u Lys Arg Leu Phe Phe                  100      - #           105      - #           110                  - - Asn Ile Ser Ala Ile Glu Lys Glu Glu Arg Va - #l Thr Met Gly Ser Gly              115          - #       120          - #       125                      - - Ile Glu Val Gln Pro Glu His Leu Leu Arg Ly - #s Gly Ile Asp Leu Arg          130              - #   135              - #   140                          - - Leu Tyr Arg Thr Leu Gln Ile Thr Leu Lys Gl - #y Met Gly Arg Ser Lys      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Thr Ser Arg Lys Leu Leu Val Ala Gln Thr Ph - #e Arg Leu Leu His        Lys                                                                                             165  - #               170  - #               175             - - Ser Leu Phe Phe Asn Leu Thr Glu Ile Cys Gl - #n Ser Trp Gln Asp Pro                  180      - #           185      - #           190                  - - Leu Lys Asn Leu Gly Leu Val Leu Glu Ile Ph - #e Pro Lys Lys Glu Ser              195          - #       200          - #       205                      - - Ser Trp Met Ser Thr Ala Asn Asp Glu Cys Ly - #s Asp Ile Gln Thr Phe          210              - #   215              - #   220                          - - Leu Tyr Thr Ser Leu Leu Thr Val Thr Leu As - #n Pro Leu Arg Cys Lys      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Arg Pro Arg Arg Lys Arg Ser Tyr Ser Lys Le - #u Pro Phe Thr Ala        Ser                                                                                             245  - #               250  - #               255             - - Asn Ile Cys Lys Lys Arg His Leu Tyr Val Gl - #u Phe Lys Asp Val Gly                  260      - #           265      - #           270                  - - Trp Gln Asn Trp Val Ile Ala Pro Gln Gly Ty - #r Met Ala Asn Tyr Cys              275          - #       280          - #       285                      - - Tyr Gly Glu Cys Pro Tyr Pro Leu Thr Glu Il - #e Leu Asn Gly Ser Asn          290              - #   295              - #   300                          - - His Ala Ile Leu Gln Thr Leu Val His Ser Il - #e Glu Pro Glu Asp Ile      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Pro Leu Pro Cys Cys Val Pro Thr Lys Met Se - #r Pro Ile Ser Met        Leu                                                                                             325  - #               330  - #               335             - - Phe Tyr Asp Asn Asn Asp Asn Val Val Leu Ar - #g His Tyr Glu Asn Met                  340      - #           345      - #           350                  - - Ala Val Asp Glu Cys Gly Cys Arg                                                  355          - #       360                                             - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - CGCGCGAAGC TTATGCTCCT GGTGTTGCTG GTG       - #                  - #             33                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - GCGCGCTCTA GATCATATGC AGTGGCAGTC TTT       - #                  - #             33                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 40 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - GCGCAGATCT GCCACCATGC TCCTGGTGTT GCTGGTGCTG     - #                      - #    40                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 61 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - CGCGAGATCT TCAAGCGTAG TCTGGGACGT CGTATGGGTA TATGCAGTGG CA -             #GTCTTTGG     60                                                                 - - C                  - #                  - #                  - #                   61                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - GCGCAGATCT GCCACCATGC TCCTGGTGTT GCT       - #                  - #             33                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - CGCGAGATCT TCATATGCAG TGGCAGTCTT TGGC       - #                  -      #        34                                                                   __________________________________________________________________________

What is claimed is:
 1. An isolated polynucleotide comprising apolynucleotide member selected from the group consisting of:(a) apolynucleotide encoding a polypeptide comprising amino acids 16 to 295of SEQ ID NO:2; and (b) the complement of (a).
 2. The isolatedpolynucleotide of claim 1 wherein said member is (a).
 3. A method ofmaking a recombinant vector comprising inserting the isolatedpolynucleotide of claim 2 into a vector.
 4. A recombinant vectorcomprising the polynucleotide of claim
 2. 5. The recombinant vector ofclaim 4 wherein said polynucleotide is operatively associated with aheterologous regulatory sequence that controls gene expression.
 6. Arecombinant host cell comprising the polynucleotide of claim
 2. 7. Aprocess for producing a polypeptide comprising:culturing a recombinantcell containing the polynucleotide of claim 2 under conditions suitableto produce the polypeptide encoded by said polynucleotide and isolatingsaid polypeptide.
 8. The isolated polynucleotide of claim 1 wherein saidmember is (a) and the polypeptide comprises amino acids 1 to 295 of SEQID NO:2.
 9. The isolated polynucleotide of claim 1 wherein said memberis (b).
 10. The isolated polynucleotide of claim 1 wherein thepolynucleotide is DNA.
 11. The isolated polynucleotide of claim 1wherein said polynucleotide is RNA.
 12. The isolated polynucleotide ofclaim 1 comprising nucleotides 67 to 948 of SEQ ID NO:1.
 13. Theisolated polynucleotide of claim 1 comprising nucleotides 64 to 951 ofSEQ ID NO:1.
 14. The isolated polynucleotide of claim 1 comprising thepolynucleotide having a polynucleotide sequence of SEQ ID NO:1.
 15. Theisolated polynucleotide of claim 1 comprising nucleotides 112 to 948 ofSEQ ID NO:1.
 16. The isolated polynucleotide of claim 1 furthercomprising a heterologous polynucleotide.
 17. The isolatedpolynucleotide of claim 16 wherein said heterologous polynucleotideencodes a heterologous polypeptide.
 18. An isolated polynucleotidecomprising a polynucleotide member selected from the group consistingof:(a) a polynucleotide encoding the same mature polypeptide encoded bythe human cDNA in ATCC Deposit No. 75902; and (b) the complement of (a).19. The isolated polynucleotide of claim 18, wherein the member is (a).20. A process for producing a polypeptide comprising:culturing arecombinant cell containing the polynucleotide of claim 19 underconditions suitable to produce the polypeptide encoded by saidpolynucleotide and isolating said polypeptide.
 21. The isolatedpolynucleotide of claim 18 wherein the member is (b).
 22. The isolatedpolynucleotide of claim 18 wherein the polynucleotide is DNA.
 23. Theisolated polynucleotide of claim 18 wherein said polynucleotide is RNA.24. A method of making a recombinant vector comprising inserting theisolated polynucleotide of claim 18 into a vector.
 25. A recombinantvector comprising the polynucleotide of claim
 18. 26. The recombinantvector of claim 25 wherein said polynucleotide is operatively associatedwith a heterologous regulatory sequence that controls gene expression.27. A recombinant host cell comprising the polynucleotide of claim 18.28. The isolated polynucleotide of claim 18 further comprising aheterologous polynucleotide.
 29. The isolated polynucleotide of claim 28wherein said heterologous polynucleotide encodes a heterologouspolypeptide.